1. Field of the Invention
The present invention relates to novel diterpenoid compounds. More particularly, it relates to novel diterpenoid compounds imparting stress resistance to plants, their uses and methods of synthesizing the same.
2. Background of the Invention
Organisms such as plants are exposed to various stresses (e.g., injury, insect pests, disease and the like) and are required to protect themselves during their lifespan. Such stresses are highly likely to cause damage which can threaten survival of plants, as compared to organisms such as animals which can move by themselves.
As used herein, “wound” refers to an injury to a plant caused by cutting, friction, compression, or being fed upon by an insect or Herviora, and physiological functions of the injured sites and broader regions are disturbed. Wounds due to worms, insects or the like are referred to as wounds due to an insect pest. In addition to wounds due to such physical injuries, wounds include those due to air pollutants, or chemical causes such as alkali, acids, heavy metals and the like, as well as those due to biological causes such as infection by pathogens including viruses, bacteria, fungi and the like. Wounds due to microorganisms such as viruses, bacteria, fungi or the like are particularly referred to as disease. Against wounds caused by such physical, chemical, or biological causes, organisms such as plants have particular defense mechanisms which have adapted themselves to the environments that they reside in.
For example, injured plants must immediately cure their wound, recover their ability to absorb water etc., from roots, and prevent invasion of pathogens from the roots. When the tissues are injured, hydrogen peroxide is generated, and lignification, suberization, and oxidative cross-linking of hydroxyproline-rich proteins are started, and cell walls are repaired and reinforced (Bradley, D. et al. Cell, 79, 21-30, 1992). This prevents moisture transpiration from the wound and also serves as a physical barrier against the invasion of pathogens. When a plant is wounded, the enzymatic activity of phenylalanine ammonia lyase, which is a rate-limiting enzyme of phenylpropanoid, is elevated and the production of polyphenol and lignin, both of which have antibiotic actions, are elevated. It is known that the plants infected with pathogens newly produce a series of proteins which are referred to as pathogenesis-related proteins (also referred to as PR proteins). Two of the PR proteins is chitinase and −1,3-glucanase and these proteins are produced when a plant is wounded. It is believed that these enzymes inhibit the growth of bacteria/fungi by degrading chitin and glucan, respectively, both of which are cell wall components of bacteria/fungi, thereby these enzymes protect the plants from infection with pathogens via the wound.
A protease inhibitor is also produced for the plants' defense against being fed by insects. Protease inhibitor is a general name for a material which inhibits an enzyme, spevifically a protease which has proteolytic activity and is also induced by infection with pathogens. The leaves of tomato and potato plants induce the production of protease inhibitor II upon being fed upon by a certain insect pest. If the insects feed on tissues comprising a large amount of protease inhibitor II, then indigestion is caused and the growth of the insects are inhibited. In addition, the plants protect themselves from insect pests by attracting a natural enemy etc. For wounds, plants have various activities including concentrating substances for the recovery from the wounds by inhibiting photosynthesis other than that for required for defense, and facilitating cell division for regeneration after the cut. Moreover, it is found that responses against the wound vary depending on factors such as the extent of the growth, the extent of the wound, the extent of the optical intensity and the like.
In this way, the injured plant generates various responses, however, it has been revealed that most of the injured plants cause physiological response by inducing expression of various genes. Of the responses, it is known that jasmonic acid (JA) is involved in the signaling system in a wound-induced response. JA treatment induces the expressions of many wound-induced responsive gene, and thus it is believed that JA is a signal transmitter of the wound-induced response (Creelman, R. A. et al. Plant Cell 9:1211-1223, 1997). JA is synthesized via oxidation of linolenic acid released from a membrane by lipoxigenase and the like, which is a result of the wounds. In animals, prostaglandin and arachidonic acid metabolites such as leukotrienes which are involved in inflammatory responses are similar to JA and the related substances in structure, and are synthesized from arachidonic acid released from the membrane by phospholipase A2. In plants as well, it is suggested that phospholipase A2 is therefore involved in a liberation of linolenic acid.
Phosphatidic acid, a membrane phospholipid, is liberated from the membrane by wounds and the expression of the wound-induced genes are induced. It is believed that this liberation results from the hydrolysis of phospholipids, which are membrane components, by phspholipase D (which is moved from the cytoplasm to the membrane by elevated-intracellular Ca2+).
When the cell walls are injured, pectin which is a major component of the middle lamella in the cell wall is degraded, and oligogalacturonic acid is generated. It is known that the pectin degrading enzyme is also induced by the wounded state.
It is known that MAP kinases, which are some of the serine/threonine type protein kinase is activated by the wounded state. This activation is found to be induced by phosphorylation. Among MAP kinases activated by wounds, two kinase a are known: WIPK (wound-induced protein kinase) which is activated at the transcription level and SIPK (salicylic acid-induced protein kinase) which is not controlled by transcription. WIPK of tobacco is also known as a regulator of JA synthesis (Seo, S. et al., Science, 270, 1988-1992, 1995). In the inflammatory response of animals, it is known that MAP kinase is involved in the activation of cytoplasmic phospholipase A2 (cPLA2) and thus it is believed that WIPK controls JA synthesis by activation of cPLA2.
Ethylene and abscisic acid (ABA), both of which are phytohormones, are believed to be signaling agents in the wound-induced response since production of these phytohormones is enhanced in the wounded state and treatments of plants with these phytohormones induces the expression of wound responsive genes. It is reported that ethylene cooperates with JA and upstream of JA signaling and ABA acts upstream of JA signaling (Dong, X: Curr. Opin. Plant Biol. 1:316-322, 1998; Pelia-Cortes, H. et al. Proc. Natl. Acad. Sci. USA, 92:3106-3114, 1995). Furthermore, ethylene has an effect of accelerating the maturation of fruits and is thus considered a maturation hormone. Also, it is also known that ethylene suppresses extension growth in stems, roots and the like, and aestivation formation, while it accelerates the auxetic growth of stems and roots, the formation of root hair, the growth of stems in a certain species, germination in certain species, epinasty, and the like. JA is also suggested to be involved in the ubiquitin-proteasome system which has various cell control functions.
Wound-induced response is characterized by a systemic response. For example, upon injuring leaves, it is found that plants induces such an wound-induced response in not only injured leaves, but also in non-injured leaves. It is thought that in tomatoes the protein systemin participates in this systemic wound-induced response. Systemin is excised from its precursor prosystemin in response to the wounds, to deliver to the entire plant body through seive tubes and induce JA synthesis.
JA is also suggested to be involved in hypersensitive reaction; HR). Hypersensitive reaction refers to a reaction in which the infected cells in the plant positively die, thereby confining the pathogen, inhibiting further pathogen growth and transition to the entire plant body, and resulting in spot formation. The hypersensitive reaction is believed to be a kind of disease resistance reaction. As a result of the hypersensitive reaction, the production of not only salicylic acid (SA), but also JA and ethylene is increased. Therefore, hypersensitive cell death means wound stress. By the hypersensitive response, pathogenesis-related proteins are induced. The pathogenesis-related proteins are grouped into acidic pathogen-related proteins and basic pathogenesis-related proteins according to their isoelectric points, the former are mainly induced by salicylic acid and the latter are mainly induced by JA. The acidic pathogenesis-related proteins are poorly induced by physical stimulation such as cutting and friction.
It is well known that prostaglandin synthesis is involved in inflammatory responses in animals and is inhibited by salicylic acid or acetyl derivatives thereof, acetyl salicylic acid (also known as aspirin). Also in plants, JA and SA inhibit each others syntheses and functions (Niki et al. (1998) Plant Cell Physiol. 39:500-507). The fact that SA and JA, both of which exhibit antagonitic actions, are generated by the hypersensitive reaction suggests that these two substances finely regulate signaling in wound-induced responses.
In this way, it is anticipated that JA and SA are known to be related to the signaling of stresses such as various wounds and that regulation of the activity of JA and SA can regulate a resistance to stresses such as wounds.
A number of wound responsive genes have been isolated. The expression of these genes is mainly regulated at the transcription level. These genes include synthetases, metabolic enzymes, control proteins, defense proteins and the like.
The mechanisms from injury to expression of the wound responsive genes is considerably different among the genes. For example, for the WIPK gene and the gene encoding 1-aminocyclopropane-t-carboxylic acid (ACC) synthetase which is involved in ethylene synthesis, the accumulation of transcription products thereof are observed in several minutes to about quarter of an hour after the wound. On the other hand, the accumulation of the transcription products of the genes for protein inhibitor II and basic pathogenesis-related protein become predominant several hours after the wound. The accumulation of JA, ethylene and ABA occur within a few or several tens of minutes after the wound. As such, it is suggested that other factors may be related to the induction of the expression in WIPK gene an the like. Further, in the induction of the expression of WIPK gene and the like, other pathways are also predicted, in addition to the pathway via JA.
Therefore, the identification of factors responsible for the regulation of WIPK and SIPK may effectively impart regulation of wound-induced responses in organisms such as plants, thus imparting stress resistance. However, heretofore, such factors have not been identified. Therefore, it is desired to seek such factors in the art.